The overall objective of this proposal is to define the cellular mechanisms culminating in lethal injury of healthy and transformed liver cells. Because of the importance of tumor necrosis factor-alpha related apoptosis inducing ligand (TRAIL) in human liver pathobiology and cancer therapy, our program is working on the cellular and subcellular mechanisms causing liver injury by this death ligand. Specifically, our long term objectives are to understand the mechanisms involved in TRAIL-mediated cell death. Based on extensive preliminary data, we propose the novel CENTRAL HYPOTHESIS that TRAIL signals cell death by triggering the lysosomal pathway of cell death. A cytotoxic pathway characterized by release of the lysosomal protease cathepsin B into the cytosol which results in cathepsin B-mediated mitochondrial dysfunction and cell death. We will now employ current and complementary, molecular, biochemical and cell biological approaches to ascertain how TRAIL triggers this unexplored pathway of apoptosis. Our proposal has three SPECIFIC AIMS. FIRST, we will directly test the hypothesis that TRAIL signals lysosomal permeabilization: a) by activating proapoptotic members of the Bcl-2 family of proteins, specifically tBid activation of Bax;and b) Bax translocation to and oligomerization within lysosomal membranes causing release of cathepsin B into the cytosol. SECOND, we will test the hypothesis that the antiapoptotic protein cFLIP, commonly overexpressed in liver cancers, prevents TRAIL-mediated lysosomal permeabilization: a) by recruiting the kinase RIP1 to the TRAIL receptor complex which in turn activates ERK1/2;and b) by ERK1/2-mediated Bid phosphorylation, an inactivating post- translational modification. FINALLY, we will test the hypothesis that TRAIL-mediated injury in the bile duct ligated mouse occurs: a) by a mechanism dependent upon natural killer (NK) and natural killer-T (NKT) cells, the major cellular sources of TRAIL in the liver;and b) by hepatocyte lysosomal permeabilization. The proposal is innovative technically and conceptually as it tests new concepts for TRAIL cytotoxicity using sophisticated technologies. The significance of the information generated is that it will provide a framework for the potential development of novel therapeutic strategies effective for both promoting TRAIL therapy of human liver cancers and attenuating TRAIL-mediated human liver injury. The grant application examines the cellular mechanisms by which TRAIL, a natural death inducing protein being developed for cancer therapy, causes cell death. We propose that TRAIL induces cell death by disrupting a key cellular organelle, referred to as the lysosome. Lysosome disruption results in the release of toxic compounds into the cell culminating in cell demise. The results of these studies are germane both to mechanisms of liver injury and the use of TRAIL as a chemotherapeutic agent.